猪圆环病毒2型TaqMan实时荧光定量PCR检测方法的建立

Abstract

Abstract: The research with porcine circovirus type 2 (PCV2) of the ORF2 capsid protein gene for the purpose, design and synthesis of specific primers and probes. Gene was amplified by PCR and cloned into the pMD18-T vector, screened positive plasmid standards. By quantitative PCR optimization of reaction conditions, establishment of a real-time detection of PCV2 TaqMan quantitative PCR method. The results showed that the method was specificity, the pig pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), swine fever virus (CSFV) and other common diseases of the original pig detection results were negative.Compared with ordinary PCR method, the sensitivity was 100 times higher, up to 100 copies/μL; for the detection of clinical samples showed that the method can effectively detect lymph nodes, lungs and other tissues of PCV2.

Key words: porcine circovirus type 2; fluorescent quantitative PCR; TaqMan probe; standard curve

CLC Number:

S852.65^＋9.2

ZENG Si-yao, ZHANG Shu-qiong, YU Shao-hua, TANG Zhi-ling, CHEN Rui-ai, LUO Man-lin. Establishment of TaqMan Real-time Quantitative PCR Method for Detection of PCV2[J]. , 2012, 39(6): 41-46.

References

1 王忠田,杨汉春,郭鑫.规模化养猪场圆环病2型感染的流行病学调查[J].中国兽医杂志,2002,38(10):3~5.
2 白兴华,原志伟,李军民,等.TaqMan荧光定量RT-PCR检测猪传染性胃肠炎病毒的研究[J].检验检疫学刊,2009,19(5):17~20.
3 乔莉娟,王立贤,颜华.猪SP2A基因荧光定量PCR检测方法的建立[J].动物医学进展,2009,30(8):24~29.
4 阳成波,印遇龙,黄瑞林,等.实时定量RT-PCR的原理及方法[J].免疫学杂志,2003,19(3):145~150.
5 李坤,温国元,罗青平,等.猪圆环病毒Ⅱ型抗体间接ELISA检测方法的建立[J].华中农业大学学报,2009,28(6):710~714.
6 张锦秀,晁生玉,丁敏,等.检测猪圆环病毒2型SYBR Green Ⅰ荧光定量PCR方法的建立[J].中国畜牧兽医,2008,35(9):30~33.
7 欧阳松应,杨冬,欧阳红生,等.实时荧光定量 PCR 技术及其应用[J].生命的化学,2004,24(1):74~76.
8 郎洪武,王力,张广川,等.猪圆环病毒分离鉴定及猪断奶后多系统衰弱综合症的诊断[J].中国兽医科技,2001,31(3):3~4.
9 侯宇.荧光定量PCR技术研究进展及其应用[J].贵州农业科学,2009,37(6):29~32.
10 郭龙军,陆月华,黄立平,等.猪圆环病毒2型Cap蛋白核定位信号区抗原表位的鉴定[J].中国农业科学,2010,43(7):1480~1486.
11 黄立平,刘长明,危艳武,等.抗猪圆环病毒2型Cap蛋白中和活性单克隆抗体的制备及鉴定[J].中国预防兽医学报,2009,31(2):132~136.
12 Allan G M,Meehan B,Todd D,et al.Novel porcine circovirus from pigs with wasting disease syndrom[J].Vet Rec,1998,142:467~468.
13 Arya M,Shergill I S,Williamson M,et al.Basic principles of realtime quantitative PCR[J].Expert Rev Mol Diagn,2005,5(2):209~219.
14 Deepak S,Kottapalli K,Rakwal R,et al.Real time PCR:revolutionizing detection and expression analysis of genes[J].Curr Genomics,2007,8(4):234~251.
15 Fenaux M,Opriessnig T,Halbur P G,et al.Two amino acid mutations in the capsid protein of type 2 porcine circovirus (PCV2) enhanced PCV2 replication in vitro and attenuated the virus in vivo[J].Journal of Virology,2004,78(24):13440~13446.
16 Kawashima H,Tsunemitsu R H,et al.Effects of dexamethasone on the pathogenesis of porcine circovirus type 2 infection in piglets[J].J Comp Path,2003,129:294~302.
17 Liu J,Chen I,Kwang J.Characterization of a previously unidentified viral protein in porcine circovirus type 2 infected cells and its role in virus induced apoptosis[J].Journal of Virology,2005,79:8262~8274.
18 Mankertz A,Domingo M,Folch J M,et al.Characterization of PCV2 isolates from Spain,Germany and France[J].Virus Research,2000,66(1):65~77.
19 Meehan B,McNeilly F,Todd D,et al.Characterization of novel circovirus DNA sassociated with wasting syndrome sin pigs[J].J Gen Virol,1997,79:2171~2179.
20 Nielsen I,Vincent A,Tner B,et al.Association of lymphopenia with porcine circovirus type 2 induced post weaning multisystemic wasting syndrome PMWS[J].Veterinary Immunology and Immunopathology,2003,92:97~111.
21 Paton D,Ibata S J.Detection of transmissible gastroenteritis virus by RT-PCR and differentiation from porcine respiratory coronavirus[J].J Vir Met,1997,66:303~309.
22 Provenzano M,Mocellin S.Complementary techniques: validation of gene expression data by quantitative real time PCR[J].Adv Exp Med Biol,2007,593:66~73.
23 Schmittgen T D,Zakrajsek B A.Effect of experimental treatment on house keeping gene expression: validation by real-time,quantitative RT-PCR[J].J Biochem Biophys Methods,2000,46(1~2):69~81.
24 Truong C,Mahe D,Blanchard P,et al.Identification of immunorelevant ORF2 epitope from porcine circovirus type 2 as a serological marker for experimental and natural infection[J].Archives of Virology,2001,146:1197~1211.
25 Valasek M A,Repa J J.The power of real-time PCR[J].Adv Physiol Educ,2005,29(3):151~159.
26 Watzinger F,Ebner K,Lion T.Detection and monitoring of virus infections by real-time PCR[J].Mol Aspects Med,2006,27(2~3):254~298.

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Establishment of TaqMan Real-time Quantitative PCR Method for Detection of PCV2

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